Many factors affect gene expression in plants and other eukaryotic organisms. Recently, small RNAs, 21-26 nucleotides, have emerged as important regulators of eukaryotic gene expression. The known small regulatory RNAs fall into two basic classes. One class of small RNAs is the short interfering RNAs (siRNAs). These play essential roles in RNA silencing, a sequence-specific RNA degradation process that is triggered by double-stranded RNA (dsRNA) (see Vance and Vaucheret (2001) Science 292:2277-2280, and Zamore (2001) Nat Struct Biol 8:746-750 for recent reviews on RNA silencing in plants and animals, respectively).
One recently identified group of small RNAs are known generically as short temporal RNAs (stRNAs) and more broadly as micro-RNAs (miRNAs). miRNAs have emerged as evolutionarily conserved, RNA-based regulators of gene expression in animals and plants. miRNAs (approx. 21 to 25 nt) arise from larger precursors with a stem loop structure that are transcribed from non-protein-coding genes. microRNAs in plants and animals function as posttranscriptional negative regulators (Bartel D (2004) Cell 116, 281-297; He L and Hannon G J (2004) Nat. Rev. Genet. 5, 522-531). Plant miRNAs target a disproportionately high number of genes with functions in developmental processes, including developmental timing, control of cell proliferation, meristem identity, and patterning. Global disruption of miRNA biogenesis or function, or disruption of specific miRNA-target interactions, generally results in developmental abnormalities (Achard P et al. (2004) Development 131, 3357-3365; Chen X (2004) Science 303, 2022-2025; Emery J F et al. (2003) Curr. Biol. 13, 1768-1774; Juarez M T et al. (2004) Nature 428, 84-88; Kidner C A and Martienssen R A (2004) Nature 428, 81-84; Laufs P et al. (2004) Development 131, 4311-4322; Mallory A C et al. (2004) Curr. Biol. 14, 1035-1046; Palatnik J F et al. (2003) Nature 425, 257-263; Tang G et al. (2003) Genes Dev. 17, 49-63; Vaucheret H et al. (2004) Genes Dev. 18, 1187-1197), indicating that miRNA-based regulation is integral to pathways governing growth and development. Plant miRNAs usually contain near-perfect complementarity with target sites, which occur most commonly in protein-coding regions of mRNAs (Llave C et al. (2002) Science 297, 2053-2056; Rhoades M W et al. (2002) Cell 110, 513-520). As a result, most plant miRNAs function like siRNAs to guide target RNA cleavage (Jones-Rhoades M W and Bartel D P (2004) Mol. Cell. 14, 787-799; Kasschau K D et al. (2003) Dev. Cell 4, 205-217). In contrast, most animal miRNAs and possibly some plant miRNAs function to repress expression at the translational or cotranslational level (Ambros V (2003) Cell 113, 673-676; Aukerman M J and Sakai H (2003) Plant Cell 15, 2730-2741; Olsen P H and Ambros V (1999) Dev. Biol. 216, 671-680; Seggerson K et al. (2002) Dev. Biol. 243, 215-225). Although many animal target mRNAs code for developmental control factors, no miRNAs or targets are conserved between plants and animals (Ambros V (2003) Cell 113, 673-676).
In plant, majority of miRNA target genes are transcription factors, which are required for meristem identity, cell division, organ separation, and organ polarity. Some miRNAs have unique tissues-specific and/or temporal expression pattern. McManus et al. (RNA 8:842-850 (2002)) also studied miRNA mimics containing 19 nucleotides of uninterrupted RNA duplex, a 12-nucleotide loop length and one asymmetric stem-loop bulge composed of a single uridine opposing a double uridine. Synthetic miRNA can either be transfected into cells or expressed in the cell under the control of an RNA polymerase III promoter and cause the decreased expression of a specific target nucleotide sequence (McManus et al. (2002) RNA 8:842-850).
The mechanism of miRNA-mediated gene silencing is only slowly becoming clearer: microRNAs form through nucleolytic maturation of genetically defined RNA precursors that adopt a self-complementary foldback structure (see Allen E. et al. (2005) Cell, Vol. 121, 207-221 and the references cited therein for details). Processing yields a duplex intermediate (miRNA/miRNA*) that ultimately provides the miRNA strand to the effector complex, termed RISC (Khvorova A et al. (2003). Cell 115, 209-216). Plants contain four DICER-LIKE (DCL) proteins, one of which (DCL1) is necessary for maturation of most or all miRNA precursors (Kurihara Y and Watanabe Y (2004) Proc. Natl. Acad. Sci. USA 101, 12753-12758). The DCL1 protein contains an RNA helicase and two RNaseIII-like domains, a central PAZ domain and C-terminal dsRNA binding motifs. HEN1 functions in miRNA biogenesis or stability by methylating the 3′-terminal residue (Yu B et al (2005) Science 307, 932-935). In Arabidopsis, HASTY (HST) provides a related function for miRNA transport (Park M Y et al. (2005) Proc. Natl. Acad. Sci. USA 102, 3691-3696). Active miRNA-containing RISC complexes in plants almost certainly contain one or more ARGONAUTE proteins, such as AGO1 (Fagard M et al. (2000). Proc. Natl. Acad. Sci. USA 97, 11650-11654; Vaucheret et al. (2004) Genes Dev 1187-1197). In addition to miRNAs, plants also produce diverse sets of endogenous siRNAs. These differ from miRNAs in that they arise from double-stranded RNA, which in interacsome cases requires the activity of RNA-dependent RNA polymerases (RDRs). Arabidopsis DCL2, DCL3, RDR1, RDR2, and RDR6 have known roles in siRNA biogenesis (Dalmay T et al. (2000). Cell 101, 543-553; Mourrain P et al. (2000). Cell 101, 533-542; Peragine, A et al. (2004) Genes Dev. 18, 2368-2379; Vazquez F et al. (2004b) Mol. Cell. 16, 69-79).
Ta-siRNAs are genetically defined at specific loci and arise by phased, DICER-LIKE processing of dsRNA formed by RDR6/SGS3 activity on RNA polymerase II transcripts. Ta-siRNAs interact with target mRNAs and guide cleavage by the same mechanism as do plant miRNAs Peragine, A et al. (2004) Genes Dev. 18, 2368-2379; Vazquez F et al. (2004b) Mol. Cell. 16, 69-79). Those ta-siRNAs regulate the accumulation of targeting mRNAs (Vazquez et al., 2004, Mol Cell 16: 69-79). ta-siRNA biogenesis is directed by certain miRNAs in Arabidopsis (Allen E et al., Keystone symposium abstract 102, Jan. 8-14, 2005, Allen E et al. (2005) Cell 121:207-221). In brief, for example Arabidopsis miR173 targets single-stranded non-coding RNA transcripts and directs biogenesis of ta-siRNA via 5′ initiation, i.e. from miR173 target site, a double-stranded RNA is produced by RdR6 (an RNA-dependent RNA polymerase) along non-coding RNA transcripts, then 7 to 8 of 21-nt phases of ta-siRNA are generated by Dicer starting from a cleavage site of miRNA and target mRNA duplex (between position 10 to 11 of miR173) in 5′ to 3′ direction. Some of ta-siRNAs initiated by miR173 target mRNAs with unknown function. In contrast, Arabidopsis miR390 targets a single-stranded non-coding RNA transcript and directs biogenesis of ta-siRNA via 3′ initiation, i.e. from miR390 target site, a double-stranded RNA is produced by RdR6 along a non-coding RNA transcript, then 7 to 8 of 21-nt phases of ta-siRNA are generated by Dicer starting from a cleavage site of miRNA andtarget duplex (between position 10 to 11 of mi390) in 3′ to 5′ direction. MiR390 target site in the non-coding RNA transcript and two 21-nt phases of ta-siRNAs, 5′D7(+) and 5′D8(+), initiated by miR390 are conserved across many plant species. These ta-siRNAs target ARF3 and ARF4 (Auxin Response Factor). These data support a model in which miRNA-directed formation of a 5′ or 3′ within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other gene expression (Allen E et al. (2005) Cell 121: 207-221 and figure below).
Plants and animals use small RNAs (microRNAs [miRNAs] and siRNAs) as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs and trans-acting (ta) siRNAs form through distinct biogenesis pathways, although they both interact with target transcripts and guide cleavage. An integrated approach to identify targets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed several new classes of small RNA-regulated genes, including conventional genes such as Argonaute2 and an E2-ubiquitin conjugating enzyme. Surprisingly, five ta-siRNA-generating transcripts were identified as targets of miR173 or miR390. Rather than functioning as negative regulators, miR173- and miR390-guided biocleavage was shown to set the 21-nucleotide phase for ta-siRNA precursor processing. These data support a model in which miRNA-guided formation of a 5′ or 3′ terminus within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other genes.
The coincident register of miRNA-guided cleavage and phased Dicer-like processing of ta-siRNA precursors support the hypothesis that miRNA targeting of primary transcripts sets the 21-nucleotide phase for accurate ta-siRNA formation. Thus, seven siR255 or related ta-siRNAs (siR850, siR289, siR752, and siR438[+]) from the three TAS1 loci are all in phase relative to the respective miR173 target sites even though they originate from different positions.
MIR390 genes, miR390 target sites and ta-siRNAs in TAS3 primary transcripts, and TAS3 ta-siRNA target sites in ARF3 and ARF4 are all conserved between monocots and dicots, indicating this pathway is at least a few hundred million years old (Allen, 2005). Allen proposes a model based on a DCL-catalyzed processing of pre-ta-siRNA duplexes, which starts from ends that are defined by miRNA-guided cleavage.
ARF3 and ARF4 transcripts are targeted by TAS3 ta-siRNAs (Allen, 2005). Thus, nearly one third of all ARF genes (23 known or predicted) are regulated by either miRNAs or ta-siRNAs. ARF10, ARF16, and ARF17 are targets of miR160, while ARF6 and ARF8 are targets of miR167 (Jones-Rhoades and Bartel (2004) Mol. Cell. 14:787-799; Kasschau et al., (2003) Dev. Cell 4:205-217). The ARF proteins are transcription factors that transduce auxin signals during growth and development (Remington et al. (2004) Plant Physiol. 135:1738-1752).
Various patent applications disclose the use of dsRNA, miRNAs and siRNAs:
WO 99/07409, describes specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. WO 99/32619 and U.S. Pat. No. 6,506,559, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes. WO 99/49029 and WO 01/70949, describe certain vector expressed siRNA molecules. WO 99/53050 describes certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. WO 00/01846, describes certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific long dsRNA molecules. WO 00/44914, and WO01/68836 describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes. WO 00/63364, and WO01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs. WO 01/29058, describes the identification of specific genes involved in dsRNA-mediated RNAi. WO 01/36646, describes certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. WO 01/42443, describes certain methods for modifying genetic characteristics of an organism using certain dsRNAs. WO 01/49844, describes specific DNA expression constructs for use in facilitating gene silencing in targeted organisms. WO 01/53475, describes certain methods for isolating a Neurospora silencing gene and uses thereof. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. WO 01/70944, describes certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. WO 01/72774, describes certain Drosophila-derived gene products that may be related to RNAi in Drosophila. WO 01/75164 describes a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications. The application reveals certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. WO 01/92513 describes certain methods for mediating gene suppression by using factors that enhance RNAi. WO 02/38805, describes certain C. elegans genes identified via RNAi. WO 02/44321 discloses that double-stranded RNA (dsRNA) 19-23 nt in length induces sequence-specific post-transcriptional gene silencing in a Drosophila in vitro system. Short interfering RNAs (siRNAs) generated by an RNaseIII-like processing reaction from long dsRNA or chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. The PCT publication also provides evidence that the direction of dsRNA processing determines whether sense or antisense-identical target RNA can be cleaved by the produced siRNP complex. WO 02/55692, WO02/55693, and EP 1144623 describe certain methods for inhibiting gene expression using dsRNA. US 2002/0086356 discloses RNA interference (RNAi) in a Drosophila in vitro system using RNA segments 21-23 nucleotides (nt) in length. The patent application publication teaches that when these 21-23 nt fragments are purified and added back to Drosophila extracts, they mediate sequence-specific RNAi in the absence of long dsRNA. The patent application publication also teaches that chemically synthesized oligonucleotides of the same or similar nature can also be used to target specific mRNAs for degradation in mammalian cells. US 2002/016216 discloses a method for attenuating expression of a target gene in cultured cells by introducing double stranded RNA (dsRNA) that comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene into the cells in an amount sufficient to attenuate expression of the target gene. WO 03/006477 discloses engineered RNA precursors that when expressed in a cell are processed by the cell to produce targeted small interfering RNAs (siRNAs) that selectively silence targeted genes (by cleaving specific mRNAs) using the cell's own RNA interference (RNAi) pathway. By introducing nucleic acid molecules that encode these engineered RNA precursors into cells in vivo with appropriate regulatory sequences, expression of the engineered RNA precursors can be selectively controlled both temporally and spatially, i.e., at particular times and/or in particular tissues, organs, or cells. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA constructs. WO 03/070918 describes methods and reagents useful in modulating gene expression. Specifically, the application described double-stranded short interfering nucleic acids (siRNA) molecule that down-regulates expression of a target gene, wherein said siRNA molecule comprises no ribonucleotides and each strand of said double-stranded siRNA comprises about 21 nucleotides. WO 04/009779 discloses engineered miRNA precursor, which are designed to produce new miRNA targeting gene-of-interest. WO 04/66183 describes the invention relates to computational methods of identifying novel microRNA (miRNA) molecules and novel targets for miRNA molecules and the microRNA molecules and targets identified by such methods. US 2004/0268441 describes microRNA precursor constructs that can be designed to modulate expression of any nucleotide sequence of interest, either an endogenous plant gene or alternatively a transgene. WO 05/019453 describes a multifunctional siRNA molecules interact with a first and a second target nucleic acid sequence, methods and reagents useful in modulating gene expression. Specifically, the invention relates to synthetic chemically modified small nucleic acid molecules. WO 05/042705 discloses computer-assisted methods of identifying, designing and synthesizing siRNA nucleotide sequences for a target mRNA sequence of a target species. WO 05/042708 discloses a method for identifying siRNA target motifs in a transcript using a position-specific score matrix approach. The invention further provides a method for designing siRNAs with higher silencing efficacy and specificity. WO 05/044981 described compounds, compositions, and methods useful for modulating gene expression using short interfering nucleic acid (siRNA) molecules.
One of the major obstacles in various field of biotechnology is the difficulty to achieve parallel suppression or silencing of multiple genes in parallel. Procedures based on chimeric antisense molecules (WO 93/23551) are inefficient. Methods based on chimeric double-stranded RNA molecules (WO 03/078629) present an improvement, but the employed DNA constructs are still somewhat laborious to obtain. There is in consequence an unfulfilled need for efficient methods and compositions to achieve gene silencing in plants, especially for two and more target genes. This goal is achieved by the present invention.